APOBEC3G (A3G) is a protein expressed in human cells that serves as an antiviral host-defense factor. While the majority of studies have suggested that A3G DNA mutagenic activity on nascent single stranded proviral DNA inhibits viral replication, recent studies suggested that circumstances might exist in permissive cells where A3G mutagenic activity may benefit the virus and promote the emergence of drug-resistant strains. The proposed research will identify small molecules for therapeutic development that are also novel research tools for addressing the urgent question of whether endogenously expressed A3G deaminase activity can be modulated to exceed a mutagenic threshold necessary for it to have antiviral activity. This is a relevant question because, in permissive cells, A3G is almost entirely 'switched off'through its interaction with cellular RNA and this low level of activity may benefit the virus. Preliminary studies have identified compounds that activate A3G deaminase activity by dissociating RNA from the enzyme and these compounds had a dose- dependent antiviral effect in permissive cells where A3G was sequestered as high molecular mass complexes. These compounds are first in their class and show that RNA inhibition of A3G is reversible in living cells. The Specific Aims for this two year R21 are: (1) conduct high throughput screening and structure activity relationship analyses for A3G activators using a unique small molecule library assembled to identify target-select hits with low cytotoxicity and antiviral activity in single round infectivity assays, (2) determine the specificity and selectivity of A3G activators for inhibiting RNA binding to A3G and quantify their effect on viral DNA load and mutagenic frequency and (3) confirm the antiviral activity and low cytotoxicity of validated hits using PBMC and live virus in 7-day spreading infectivity assays. Compounds identified through this proposal achieve the goal of characterizing a novel class of compounds 'as unique research molecular probes'and that offer a new therapeutic target and drug discovery opportunity for attacking viral resistance while satisfying an unmet need for novel research tools to address controversies in the field. PUBLIC HEALTH RELEVANCE: This proposal is in response to Program Announcement PA-10-069, NIH Exploratory Developmental Research Grant Program. The ultimate goal of this proposal is to identify one or more novel antiviral compounds for therapeutic development that have nanomolar efficacy and low toxicity whose mechanism of action is to liberate APOBEC3G (A3G) from aggregates that form with host cell RNA. Such 'A3G Activators'have been identified and validated through the preliminary high throughput screening HTS with an innovative quenched FRET assay. Compounds identified through the proposed research will also serve to address an unmet need for novel research reagents to address controversy over whether A3G mutagenic activity promotes host defense or benefits the virus. This proposal is innovative because it seeks unconventional solutions for reducing viral infectivity and the emergence of viral resistance. 'The long term goal of the proposed research is to identify novel compounds that can be developed into drugs used in the treatment or prevention of HIV/AIDS'.